Simultaneous Detection of Multiplex-Amplified Human Immunodeficiency Virus Type 1 RNA, Hepatitis C Virus RNA, and Hepatitis B Virus DNA Using a Flow Cytometer Microsphere-Based Hybridization Assay

Abstract

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from thegag region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5′ noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

Quantification of hepatitis B virus genomes and infectivity in human serum samples

“…The exact copy numbers of HBV DNA per CID 50 for these inocula were not reported . Although many techniques for quantification of HBV DNA have been reported, [22][23][24][25][26][27][28][29][30][31][32] real-time PCR has been most widely used and considered to be most sensitive. [33][34][35][36][37][38][39][40] The TaqMan assay with the fluorescent probe required that the probe must completely match the target sequence; at most only one nucleotide mismatch could be tolerated.…”

Virological and Immunological Outcomes of Coinfections

“…Chemiluminescence and magnetic separation. Detection of a limited number of fluorophores, which is a drawback of qRT-PCR, may be overcome by employing a chemiluminescent label-based assay (83,84). Optical labels such as colorimetric nanoparticles (85)(86)(87), fluorescent tags (88), and chemiluminescent labels (89) are increasingly being used for DNA hybridization assays.…”

Persistent Hepatitis B Virus Infection in Subjects Without Hepatitis B Surface Antigen: Clinically Significant or Purely “Occult”?

“…We should, besides research programs, consider the present cost and technical limitations of the assay when searching for low HBV viremia and the uncertainties regarding the HBV pathogenic effects in several clinical conditions. [64][65][66] Thus, an HBV DNA test should be only performed in HBsAg-negative patients with continuing chronic hepatitic inflammation, when no other etiologic factors have been identified, and not in those with normal liver tests. HBV-DNA detection should be considered in the transplantation context in anti-HBc-positive individuals with normal liver tests evaluated for bone marrow or kidney donation.…”